In situ rna analysis using probe pair ligation

Abstract

The present invention relates to the field of ribonucleotide analysis. More specifically, the present invention provides compositions and methods for detection for nucleic acids using probe pair litigation. In particular embodiments, the compositions and methods of the present invention utilize a probe set comprising (1) a first multi-partite probe comprising a 5′ phosphorylated donor probe and a first bridge probe, wherein the 5′ phosphorylated donor probe specifically hybridizes to a target nucleic acid; and (ii) a second multi-partite probe comprising a 3′ acceptor probe and a second bridge probe, wherein the 3′ acceptor probe specifically hybridizes to the target nucleic acid adjacent to the 5′ donor probe and the second bridge probe is 5′ phosphorylated.

1 . A method for detecting an immobilized target ribonucleic acid (RNA) comprising the steps of:
 contacting a biological sample comprising the target RNA in a reaction mixture with at least one probe set comprising (i) a first multi-partite probe comprising a 5′ phosphorylated donor probe and a first bridge probe, wherein the 5′ phosphorylated donor probe specifically hybridizes to the target RNA; and (ii) a second multi-partite probe comprising a 3′ acceptor probe and a second bridge probe, wherein the 3′ acceptor probe specifically hybridizes to the target RNA adjacent to the 5′ donor probe and the second bridge probe is 5′ phosphorylated;   b. incubating the reaction mixture of step (a) under conditions that permit hybridization of the at least one probe set to the target RNA present in the biological sample;   c. washing away unbound probe sets;   d, ligating the 5′ phosphorylated donor probe and the 3′ acceptor probe;   e. contacting the reaction mixture with at least one bridge primer that specifically hybridizes to the first bridge probe and the second bridge probe, wherein the first bridge probe and the second bridge probe anneal to the bridge primer adjacent to each other;   f. ligating the first bridge probe and the second bridge probe thereby forming a circularized probe that is hybridized to the target RNA;   g. amplifying the circularized probe by rolling circle amplification; and   h. detecting the target RNA.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is fixed. 
     
     
         3 . The method of  claim 2 , wherein the fixed biological sample comprises fixed tissue, frozen-fixed tissue, formalin fixed paraffin embedded tissue, adherent fixed cells, suspension fixed cells or fixed cells. 
     
     
         4 . The method of  claim 1 , wherein the target RNA is immobilized by capture prior to step (c). 
     
     
         5 . The method of  claim 1 , wherein the 3′ acceptor probe comprises at least one 3′ terminal ribonucleotide. 
     
     
         6 . The method of  claim 1 , wherein step (e) is performed prior to step (d). 
     
     
         7 . The method of  claim 6 , wherein ligating steps (d) and (f) are performed simultaneously. 
     
     
         8 . The method of  claim 1 , wherein steps (e) and (f) are performed prior to step (d). 
     
     
         9 . The method of  claim 1 , wherein ligating step (d) is performed using a ligase selected from the group consisting of T4 RNA Ligase 2 (Rnl2), a Chlorella virus DNA ligase (PBCV-1 DNA Ligase), a T4 DNA Ligase, derivatives thereof, and combinations thereof. 
     
     
         10 . The method of  claim 1 , wherein detecting step (h) comprises sequencing of rolling circle amplification products. 
     
     
         11 . The method of  claim 10 , wherein the probe set comprises a barcode unique to the target RNA and wherein sequencing of the barcode detects the target RNA. 
     
     
         12 . The method of  claim 11 , wherein the sequencing comprises sequencing by synthesis or sequencing by ligation. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein detecting step (h) comprises sequencing the ligated sequence formed by the donor probe and the acceptor probe. 
     
     
         16 . The method of  claim 1 , wherein detecting step (h) comprises contacting the reaction mixture with a detectably labeled detector probe that specifically hybridizes the ligated sequence formed by the donor probe and the acceptor probe. 
     
     
         17 . The method of  claim 1 , wherein the first multi-partite probe and the second multi-partitie probe each further comprise at least one detection probe. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 18 , where the first multi-partite probe and the second multi-partite probe each comprise a spacer sequence between the two detection probes. 
     
     
         20 . The method of  claim 17 , wherein detecting step (h) comprises contacting the reaction mixture with at least one detectably labeled detector probe that specifically hybridizes to the at least one detection probe and imaging the at least one detectable labeled detector probe. 
     
     
         21 . The method of  claim 20 , further comprising the step of identifying the location of the target RNA in the sample. 
     
     
         22 . The method of  claim 20 , further comprising the step of quantifying the target RNA in the sample. 
     
     
         23 .- 27 . (canceled) 
     
     
         28 . A kit comprising:
 a probe set comprising (i) a first multi-partite probe comprising a 5′ phosphorylated donor probe and a first bridge probe, wherein the 5′ phosphorylated donor probe specifically hybridizes to a target RNA; and (ii) a second multi-partite probe comprising a 3′ acceptor probe and a second bridge probe, wherein the 3′ acceptor probe specifically hybridizes to the target RNA adjacent to the 5′ donor probe and the second bridge probe is 5′ phosphorylated.   
     
     
         29 .- 41 . (canceled)